Our previous studies have demonstrated the in-vitro and in-vivo targeting of a generation-5 (G5) dendrimer-based multifunctional conjugate, which used folic acid (FA) as the targeting agent and methotrexate (MTX) as the chemotherapeutic drug. For the synthesized G5-FA-MTX nanodevice conjugate to be clinically applicable as a cancer therapeutic drug, it is important that the compound elicits cytotoxicity specifically and consistently. The aim of this work was to evaluate four independently synthesized batches of G5-FA-MTX conjugates for their cytotoxic potential and specificity. For determination of specificity, we have used a unique 'coculture' assay in which FA receptor-positive and FA receptor-negative cells were cultured together and have examined the preferential killing of the former. The results of our study show the batch-to-batch consistency and specificity of the G5-FA-MTX nanodevice in the preferential killing of FA receptor-positive cells. The coculture assay shows the consistency of the four different G5-FA-MTX conjugate lots in the specific killing of targeted cells. Further in-vivo studies are, however, necessary to prove the clinical potential of this targeted therapeutic nanodevice.

Locally advanced rectal cancers are treated with neoadjuvant chemoradiation therapy followed by surgery. In a minority (∽20%) of patients, no tumor is present at the time of surgery; these patients with a complete pathologic response (pathCR) to neoadjuvant therapy have better treatment outcomes. Unfortunately, the inherent radioresistance of colorectal cancer (CRC) cells dictates that the majority of patients do not achieve a pathCR. Efforts to improve these odds have fueled the search for novel, relatively less-toxic radiosensitizers with distinct molecular mechanism(s) and broad-spectrum anticancer activities. Here, we use zerumbone, a sesquiterpene from the edible ginger (Zingiber zerumbet Smith), to enhance radiosensitivity of CRC cells. Short exposure to zerumbone (7 h) profoundly sensitized CRC cells, independent of their p53 or k-RAS status. Zerumbone enhanced radiation-induced cell cycle arrest (G2/M), increased radiation-induced apoptosis, but induced little apoptosis by itself. Zerumbone significantly enhanced radiation-induced DNA damage, as evident by delayed resolution of post-irradiation nuclear γH2AX foci, whereas zerumbone treatment alone did not induce γH2AX foci formation. Zerumbone pretreatment inhibited radiation-induced nuclear expression of DNA repair proteins ataxia-telangiectasia mutated (ATM) and DNA-PKcs. Interestingly, zerumbone-mediated radiosensitization did not involve reactive oxygen species (ROS), but was mediated through depletion of cellular glutathione (GSH). Ability of only thiol-based antioxidants to abrogate zerumbone-mediated radiosensitization further corroborated this hypothesis. The α,β-unsaturated carbonyl group in zerumbone was found to be essential for its bioactivity as zerumbone analog α-Humulene that lacks this functional group, could neither radiosensitize CRC cells, nor deplete cellular GSH. Our studies elucidate novel mechanism(s) of zerumbone's ability to enhance CRC radiosensitivity.

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